Correlation of N-acetyltransferase 2 genotype and acetylation status with plasma isoniazid concentration and its metabolic ratio in ethiopian tuberculosis patients

Unfavorable treatment outcomes for tuberculosis (TB) treatment might result from altered plasma exposure to antitubercular drugs in TB patients. The present study investigated the distribution of the N-Acetyltransferase 2 (NAT2) genotype, isoniazid acetylation status, genotype–phenotype concordance of NAT2, and isoniazid plasma exposure among Ethiopian tuberculosis patients. Blood samples were collected from newly diagnosed TB patients receiving a fixed dose combination of first-line antitubercular drugs daily. Genotyping of NAT2 was done using TaqMan drug metabolism assay. Isoniazid and its metabolite concentration were determined using validated liquid chromatography-tandem mass spectrometry (LC–MS/MS). A total of 120 patients (63 male and 57 female) were enrolled in this study. The mean daily dose of isoniazid was 4.71 mg/kg. The frequency of slow, intermediate, and fast NAT2 acetylators genotypes were 74.2%, 22.4%, and 3.3% respectively. The overall median isoniazid maximum plasma concentration (Cmax) was 4.77 µg/mL and the AUC0–7 h was 11.21 µg.h/mL. The median Cmax in slow, intermediate, and fast acetylators were 5.65, 3.44, and 2.47 μg/mL, respectively. The median AUC0–7 h hour in slow, intermediate, and fast acetylators were 13.1, 6.086, and 3.73 mg•h/L, respectively. The majority (87.5%) of the study participants achieved isoniazid Cmax of above 3 µg/mL, which is considered a lower limit for a favorable treatment outcome. There is 85% concordance between the NAT2 genotype and acetylation phenotypes. NAT2 genotype, female sex, and dose were independent predictors of Cmax and AUC0–7 h (p < 0.001). Our finding revealed that there is a high frequency of slow NAT2 genotypes. The plasma Cmax of isoniazid was higher in the female and slow acetylators genotype group. The overall target plasma isoniazid concentrations in Ethiopian tuberculosis patients were achieved in the majority of the patients. Therefore, it is important to monitor adverse drug reactions and the use of a higher dose of isoniazid should be closely monitored.

www.nature.com/scientificreports/ The introduction of isoniazid, which is relatively inexpensive, and well tolerated in 1952, for tuberculosis care opened the modern era of tuberculosis treatment 7 . Isoniazid has high early bactericidal activity (EBA) and it can reduce bacterial load by 90-95% in the first 2 days of treatment 8 . EBA activity of isoniazid depends on the concentration that reaches the bacilli 9 . Several pharmacokinetic studies suggest a target of 3-6 μg/mL for the peak concentration (C max ) following a 300 mg once-daily dose of isoniazid. The C max of isoniazid occurs 1-2 h post-dose 10 .
Isoniazid is primarily metabolized to N-acetyl-isoniazid by the arylamine N-acetyltransferase 2 (NAT2) enzyme 11 . The NAT2 gene is located on chromosome 8p22 and encodes the NAT2 enzyme 12 . NAT2 gene is highly polymorphic displaying wide between-patient and between-population variations in its expression and enzyme activity. NAT2*4 is a wild-type allele which is a fast acetylator genotype, while the common defective variant alleles (NAT2*5, *6, *7, and *14) result in decreased acetylation activity and slow acetylation status. Intermediate acetylators carry one copy of NAT2*4 13 . Identification of NAT2 polymorphism is useful to predict the effective therapeutic doses and adverse effects of isoniazid in different acetylators groups 14,15 . Slow acetylators are at increased risk of toxicity from isoniazid 16,17 while fast acetylators are at increased risk of treatment failure 6 .
Isoniazid is metabolized to N-acetylisoniazid (AcINH) by the NAT2 enzyme, isonicotinic acid (INA), and hydrazine (Hz) by the amidase enzyme. NAT2 also catalyzes the acetylation of acetyl hydrazine (AcHz), which is a metabolite of AcINH, to non-toxic diacetylhydrazine. It also undergoes non-enzymatic conjugation with various endogenous substrates such as vitamin B6 18 . The mechanism by which isoniazid induces liver injury is not well established but believed that the metabolism of isoniazid produces a reactive metabolite that causes liver damage. Nevertheless, several recent studies showed that slow acetylators are at increased risk of hepatotoxicity 14,[16][17][18] .
The distribution of slow acetylator and NAT-2 defective variant allele frequency varies across regions and populations within the region 19,20 . Black Africans display wide variations in NAT2 genotype frequencies and slow acetylator phenotypes than non-Africans. Similarly, previous studies reported a high frequency of slow acetylator genotypes and phenotypes in the Ethiopian population 21,22 . Higher plasma isoniazid concentration was observed in Ethiopian pediatric patients 21 . On another hand, sub-clinical hepatotoxicity was observed in 17.3% of the patients who received the first-line antitubercular drugs 23 .
Ethiopia is listed among the top 20 high TB and TB/HIV burden countries globally 24 . Isoniazid is part of the first-line anti-TB regimen in the country. Variations in the isoniazid acetylation rate, partly due to NAT2 genetic variation, may influence TB treatment outcomes. But data is lacking on the distribution of the NAT2 acetylation status and its relationship with plasma isoniazid concentrations among Ethiopian TB patients. Therefore, this study investigated the distribution of the NAT2 genotype-based acetylation status and its correlation with the C max and plasma exposure (AUC 0-7 h ) of isoniazid in Ethiopian tuberculosis patients.

Materials and methods
Study participants. The study population comprised adult TB patients aged 18-65 years, receiving standard first-line drugs for TB treatment according to the Ethiopian treatment guidelines 25 . Newly diagnosed patients with drug-susceptible Mycobacterium TB were recruited from the TB clinics of the health center found in Addis Ababa (Beletshachew, Teklehymanote, Kazanchis, Woreda 2, and Areda Health Centre) from October 2019 to November 2021. Patients with either pulmonary or extrapulmonary forms of TB were included in the study. Patients received a daily dose of fixed-dose combination tablets containing 150, 75, 400, and 275 mg of rifampicin, isoniazid, pyrazinamide, and ethambutol respectively. The number of tablets received daily was based on the patient's body weight. Patients with a body weight greater than 55 kg received four fixed-dose combinations (FDC) tablets daily. Patients with a body weight between 40 and 55 kg received three FDC tablets daily and those under 40 received two FDC tablets. Treatment was provided under directly observed therapy (DOTs) at a primary health care facility found in Addis Ababa.
The study received ethical approval from the Institutional Review Board of the College of Health Science, Addis Ababa University (Ref number 080/17/IM), and the national research ethics review committee (Ref. Number MoSHE/RD/401/10,975/20). The study was conducted following the ethical principle of the Helsinki Declaration. All participants received a detailed explanation of the study protocol and provided written informed consent.
Blood sample collection. Blood samples were collected after observing drug intake in an EDTA tube. The sample was collected 2 weeks post-treatment initiation and only during the intensive phases of treatment. Blood samples were drawn at three-time points ranging from 1 to 7 h post-drug intake. But for a few patients, blood samples were drawn at two-time points. Plasma was separated immediately and stored at − 80 °C at the Department of Pharmacology and Clinical Pharmacy at Addis Ababa University until being transported to Karolinska Institutet in Stockholm, Sweden for analysis on dry ice. DNA extraction and SNP genotyping. Genomic DNA was extracted from whole blood samples using the QIAmp DNA Blood Midi Kit (QIAGEN GmbH, Hilden, Germany) following the manufacturer's protocol. DNA was quantified using a NanoDrop spectrophotometer (Thermo Scientific) and stored at -20 °C until genotyping assay analysis. The recommended 4-SNP genotype panel of NAT2*5 (c.341 T > C), NAT2*6 (c.590G > A), NAT2*7 (c.857G > A), NAT2*14 (191G > A, rs1801279) for reliable estimation of rapid, intermediate, and slow acetylator phenotypes were selected 13,26 . Genotyping was performed using TaqMan drug metabolism assay reagents for allelic discrimination (Applied Biosystems Genotyping Assays) with the following ID numbers for each SNP: C___1204093_20 for NAT2*5 (c.341 T > C, rs1801280), C___1204091_10 for NAT2*6 (c.590G > A, rs1799930), C____572770_20 for NAT2*7 (c.857G > A, rs1799931), C____572770_20 for NAT2*14 (191G > A, rs1801279). The final volume for each reaction was 10 μL, consisting of 9 μL TaqMan® fast advanced master

Quantification of plasma isoniazid and its metabolite concentration.
For the determination of plasma concentration of isoniazid 4 mL venous blood was collected 2 weeks post-treatment initiation in the morning after an overnight fast in an EDTA tube. Plasma was separated immediately and stored at − 80 °C at the department of pharmacology and clinical pharmacy, Addis Ababa University until transported to Karolinska Institutet, Stockholm, Sweden for analysis. Quantification of isoniazid and acetyl-isoniazid were done at the therapeutic drug monitoring laboratory, Department of Clinical Pharmacology, Karolinska University Hospital. In brief, the concentration of isoniazid and acetyl-isoniazid were determined simultaneously using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) system consisting of an Acquity Ultra Performance LC-system coupled to a Xevo TQ-S Micro (Waters, Milford, MA, USA). The chromatographic column consisted of YMC-ultraHT hydrosphere C18, 2 μm, 100 × 2 mm, reversed-phase column (Waters). And the mobile phase gradient of 0.1% formic acid in Milli-Q pure water, 100% methanol: methanol/Milli-Q pure water: Formic acid (10:90:0.1), methanol: Milli-Q pure water: isopropanol: Formic acid (70: 20: 10: 0.1), Methanol: Milli-Q pure water (10:90). The plasma sample preparation was based on protein precipitation with acetonitrile containing Isoniazid-d4, and Acetylisoniazid-d4 as an internal standard. The lower limit of quantification for isoniazid and acetyl-isoniazid were 0.05 µg/mL and 0.05 µg/mL respectively and the quantification ranges were 0.05-20 µg/ mL and 0.05-10 µg/mL respectively. The method was validated according to the European Medicines Agency Guideline on bioanalytical criteria 27 .

Statistical analysis.
For each patient, the C max was defined as the highest concentration measured, and the Tmax was the time point at which the C max occurred. AUC 0-7 h calculation was performed using the trapezoidal rule. Graphpad prism was used to calculate AUC 0-7 h . Continuous data were presented as median (interquartile range) for non-normal distributed data and mean standard deviation for normally distributed data. The Chi-square test was used to assess the Hardy-Weinberg equilibrium and genotype-phenotype concordance. Kruskal-Wallis tests were performed to see differences in C max of isoniazid and acetyl isoniazid concentrations among the different genotypes. Univariate followed by stepwise multivariate linear regression analysis was performed to identify a predictive factor of isoniazid C max and AUC 0-7 h . Statistical analyses were performed using SPSS, version 27. P value < 0.05 was considered statistically significant.
Isoniazid plasma exposure. Spare pharmacokinetic sampling during the intensive phase of the therapy was done (median sampling point = 20 days after anti-TB treatment initiation, range = 11 to 46 days). Plasma sampling took place three times for 112 (92.5%) patients, two times for 7 (5.8%) patients, and one time for 1 (0.8%) patient. Plasma sampling time ranges from 1 to 7 h post-drug intake on an empty stomach. C max was determined by taking the highest of the measured isoniazid plasma concentration. The time at which Cmax was observed is shown in Fig. 1. The regression line in Fig. 1 shows that the highest Cmax is achieved when the plasma is sampled earlier and gradually decreases as the time of sampling increases.
Isoniazid plasma exposure displayed wide between-patient variability, with the median C max being 4.77 µg/ mL (IQR 3.78-5.96). A comparison of the median C max of isoniazid (Fig. 2), isoniazid AUC 0-7 h , and acetyl isoniazid between fast, intermediate, and slow acetylators is shown in Table 3. There was a significant difference in There was a significant difference in isoniazid AUC 0-7 h between acetylator groups. The overall median isoniazid AUC 0-7 h for slow, intermediate, and fast acetylators was 13.09 µg.h/mL, 6.09 µg.h/mL, and 3.73 µg.h/ mL, respectively. The variation of AUC 0-7 h between the slow genotype group and the other two groups is high (p < 0.001). Similarly, acetyl-isoniazid C max concentration varies among the three NAT2 genotypes. A significant difference in acetyl-isoniazid concentration was observed between slow and intermediate (p < 0.001) and slow    Isoniazid metabolic ratio. Plasma acetyl-isoniazid to isoniazid ratio (AcINH/INH) ranged between 0.01 to 2.24 (median = 0.145, IQR = 0.106-0.295). The classification of acetylator phenotypes as slow and fast was done as described by Varshney E et al. 28 and Aklillu et al. 22 . In brief, a probit plot and regression analysis were used to identify the anti-mode cut-off value to classify slow and rapid acetylators. The cut-off value identified for AcINH/INH ratio was 0.473 and according to this cut-off value, 86.3% of study participants were classified phenotypically as slow acetylators and the remaining 12.76% as fast acetylators.  www.nature.com/scientificreports/ NAT2 genotype-phenotype concordance. The phenotype-genotype concordance was described using traditional phenotype classification. Genotype inferred acetylations status described above. Similarly, using AcINH/INH ratio phenotypic acetylation status as slow and fast acetylators was done as described above.

Discussion
The effect of NAT2 genotype on the pharmacokinetics of isoniazid in TB patients is well explored in various Asian and Caucasian populations but data is scarce from sub-Sharan Africa, including Ethiopia, the seventh top high-TB burden country globally and the 2nd most populous nation in Africa. Ethiopians display wide pharmacogenetics variations compared to other populations within and outside of Africa 29,30 . In this study, we investigated the profile and predictors of isoniazid plasma exposure and the effect of the NAT2 genotype on isoniazid and its metabolite acetyl isoniazid pharmacokinetics in a cohort of newly diagnosed Ethiopian tuberculosis patients. Our main findings include i) a significant association of NAT2 acetylator genotype with between-patient variability in isoniazid pharmacokinetics (C max , AUC 0-7 h , metabolic ratio), ii) a high concordance rate (85%) between NAT2 genotype and acetylation rate of isoniazid, iii) high prevalence of slow acetylators in Ethiopian TB patients and the majority of (85%) achieved therapeutic isoniazid plasma concentration. iv) NAT2 genotype and sex are significant predictors of isoniazid plasma exposure. Interestingly, we found a high prevalence of genotypic (74.2%) and phenotypic (86%) slow acetylators in Ethiopian TB patients. Genotypically, 22.4% were intermediate acetylators, and only 3.3% were fast acetylators. Our finding is in line with a previous study among healthy Ethiopians, reporting the frequency of slow, intermediate, and fast acetylators being 73.6%, 24.6%, and 1.8%, respectively 22 . The frequency distribution of the slow acetylators genotype varies between populations. About 10-20% of Asians and 40-70% of Caucasians are slow acetylators 31 . Black Africans, the most genetically diverse population on earth, display the highest level of within-population diversity of NAT2 genotype and outside of the region 19,20 . The fast acetylators are predominant in West Africa. Compared to the Ethiopians, a lower frequency of slow acetylators in Senegalese (44.3%) 32 , South African (52.5%) 33 , and Tanzanians (48%) TB patients 34 is reported. This confirms the wide heterogeneity of black Africans and results from one population may not apply to others within the region.
Various levels of concordance between the NAT2 genotype and acetylation phenotype are reported. Our study revealed high concordance (85%) between NAT 2 genotype and NAT2 acetylation phenotype. Aklillu et.al 22 reported a lower (75%) but significant NAT2 genotype-phenotype concordance in healthy Ethiopians using caffeine as a probe drug for NAT2 enzyme activity. Unlike our finding in Ethiopian TB patients, a recent study in Zulu-speaking South Africans reported a lower percentage (55%) of slow acetylators and poor or no significant concordance between the NAT2 genotype and isoniazid phenotype concordance 33 .
Low isoniazid concentrations have been postulated to result in unfavorable treatment outcomes 35,36 . A target of 3-6 μg/mL for the peak concentration (C max ) following a 300-mg daily dose of isoniazid is considered vital for a favorable treatment outcome 35 . Studies also reported that anti-TB drug-induced hepatotoxicity was associated with slow acetylation 37 . In this study, the C max of isoniazid was greater than 3 µg/mL in 87.5% of patients and the AUC 0-7 h of isoniazid was high suggesting high isoniazid exposure in Ethiopian tuberculosis patients. A similar pattern of isoniazid plasma concentration was observed in Ethiopian pediatric TB patients 21 . The large proportion of slow acetylators in our cohort means isoniazid plasma exposure is sufficiently high to provide clinical benefit.
In univariate and multivariate analysis, sex, and NAT2 acetylator genotype status were predictors of isoniazid C max . Females had higher C max compared to males, which is in agreement with those of previous studies 38, 39 . This may explain the previous finding of an increased risk of isoniazid-induced drug toxicity 40 and a lower risk of unfavorable treatment outcomes in females 41 . Plasma isoniazid C max increased as the isoniazid dose increased. This suggests that dose is also a predictor of C max . Nonsmokers had higher isoniazid C max than smokers; inversely khat chewer had higher isoniazid AUC 0-7 h than nonchewers though the differences in both were not significant. www.nature.com/scientificreports/ NAT2 enzyme activity is the rate-limiting step in acetylating isoniazid to acetyl isoniazid. A high interindividual variation was observed in the clinical efficacy, elimination, and side effects of isoniazid. These variations were related to the difference in the NAT2 enzyme which metabolizes isoniazid. We observed a bimodal isoniazid C max , unlike other studies which reported trimodal C max and AUC based on NAT2 genotype 42,43 . A significant variation of isoniazid C max and AUC 0-7 were observed between the slow and the other two acetylators groups while there is no significant variation between fast and intermediate acetylators. The low number of fast acetylators in our study population might be attributed to the absence of difference between fast and intermediate acetylators groups. Fast isoniazid acetylators showed lower C max and exposure to the drug than slow acetylators. Several authors reported an increased risk of toxicity in slow acetylators 37,44 and an increased risk of therapeutic failure in fast acetylators 34,45 patients. NAT2 genotype-guided isoniazid administration reduced toxicity and improved treatment outcomes in Japanese trials 46 . Thus, owing to high exposure to isoniazid, Ethiopians are at increased risk of toxicity from isoniazid. Indeed high rates of anti-TB and antiretroviral treatment-induced liver toxicity in Ethiopian TB-HIV coinfected patients, particularly in slow acetylators is reported previously 47 .
We evaluated the plasma isoniazid C max and drug exposure following standard laboratory recommendations like collecting plasma from patients who have received anti-drug after fasting overnight. Several studies reported that food decreased absolute bioavailability and maximum concentration of isoniazid 48,49 . Plasma was immediately separated and kept at -20 °C until transported for storage at − 80 °C on the same day. The cold chain was kept during sample transportation. The concentration of isoniazid after a week of storage at -20 °C was about 80% of the initial amount and no significant change in the initial concentration was observed if stored at -80 °C for longer than six months 50 . Study participants were patients receiving a standard dose of isoniazid in a fixed dose combination with rifampicin, ethambutol, and pyrazinamide. Patients had no prior exposure to the drugs and had no reported comorbidities of liver, kidney, HIV infection, or diabetes. Low isoniazid concentration was observed in TB-HIV co-infected patients 51 .
Our study has some limitations. Although the spare sampling strategy is evolving in recent years and found to be useful to capture AUC0-24 h 52 , the time point at which we collected the plasma sample varied from patient to patient. The sparse sampling strategy may not fully define the individual C max and AUC. We enrolled 120 patients in both the pharmacokinetics and pharmacogenetics studies. Because of the low frequency of fast acetylators in our study participants, we did not observe significant pharmacokinetics variation between fast and intermediate acetylators.
In conclusion, we report a high prevalence of the slow NAT2 acetylator genotype in Ethiopian tuberculosis patients. NAT2 acetylation status and the female sex are strong predictors of isoniazid plasma concentrations. The majority of the patients attain therapeutic plasma isoniazid exposure for a favorable treatment outcome. On the other hand, slow acetylators and females are at a higher risk of concentration-dependent isoniazid toxicity. Therefore, close safety monitoring, particularly for patients on high-dose isoniazid short-course MDR-TB therapy is recommended for early identification and management of treatment-associated adverse events.

Data availability
All data generated or analyzed in this study are included in this article. The datasets used and/or analyzed during the study are available from the corresponding author upon reasonable request.